WO2005108561A2 - Enzyme recombinante presentant une sensibilite modifiee a la reaction - Google Patents

Enzyme recombinante presentant une sensibilite modifiee a la reaction Download PDF

Info

Publication number
WO2005108561A2
WO2005108561A2 PCT/EP2005/052180 EP2005052180W WO2005108561A2 WO 2005108561 A2 WO2005108561 A2 WO 2005108561A2 EP 2005052180 W EP2005052180 W EP 2005052180W WO 2005108561 A2 WO2005108561 A2 WO 2005108561A2
Authority
WO
WIPO (PCT)
Prior art keywords
preferentially
conserved region
conserved
amino acid
modified
Prior art date
Application number
PCT/EP2005/052180
Other languages
English (en)
Other versions
WO2005108561A3 (fr
Inventor
Gwénaëlle BESTEL-CORRE
Michel Chateau
Rainer Martin Figge
Céline RAYNAUD
Philippe Noel Paul Soucaille
Original Assignee
Metabolic Explorer
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Metabolic Explorer filed Critical Metabolic Explorer
Priority to PL05742717T priority Critical patent/PL1747269T3/pl
Priority to JP2007512216A priority patent/JP5164566B2/ja
Priority to CN2005800153152A priority patent/CN1954067B/zh
Priority to US11/579,907 priority patent/US8795990B2/en
Priority to BRPI0510819-5A priority patent/BRPI0510819B1/pt
Priority to MXPA06013125A priority patent/MXPA06013125A/es
Priority to ES05742717.1T priority patent/ES2535816T3/es
Priority to EP05742717.1A priority patent/EP1747269B1/fr
Publication of WO2005108561A2 publication Critical patent/WO2005108561A2/fr
Publication of WO2005108561A3 publication Critical patent/WO2005108561A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

Definitions

  • the present invention relates to the use of recombinant homoserine transsuccinylase enzymes with altered feedback sensitivity (MetA*) and eventually, recombinant S-adenosyl methionine synthetase enzymes with reduced activity (MetK*) for the production of methionine, its precursors or derivatives thereof.
  • Sulfur-containing compounds such as cysteine, homocysteine, methionine or S- adenosylmethionine are critical to cellular metabolism and are produced industrially to be used as food or feed additives and pharmaceuticals.
  • methionine an essential amino acid, which cannot be synthesized by animals, plays an important role in many body functions. Aside from its role in protein biosynthesis, methionine is involved in transmethylation and in the bioavailability of selenium and zinc. Methionine is also directly used as a treatment for disorders like allergy and rheumatic fever. Nevertheless most of the methionine which is produced is added to animal feed.
  • Methionine is derived from the amino acid aspartate, but its synthesis requires the convergence of two additional pathways, cysteine biosynthesis and Cl metabolism (N- methyltetrahydrofolale). Aspartate is converted into homoserine by a sequence of three reactions. Homoserine can subsequently enter the threonine/isoleucine or methionine biosynthetic pathway. In F.. coli entry into the methionine pathway requires the acylation of homoserine to succinyl- homoserine. This activation step allows subsequent condensation with cysteine, leading to the thioether-containing cystadiionine, which is hydrolyzed to give homocysteine. The final methyl transfer leading to methionine is carried out by either a Bi 2 -dcpcndent or a B ]2 -indcpcndcnt methyltransferase.
  • Methionine biosynthesis in E. coli is regulated by repression and activation of methionine biosynthetic genes via the MeU and MetR proteins, respectively (reviewed in Neidhardt, F. C. (Ed. in Chief), R. Curliss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Rilcy, M. Schaechtcr, and H. E. Umbargcr (eds). 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology; Weissbach et al., 1991 Mol. Microbiol., 5, 1593-1597).
  • MetJ uses S-adenosylmethionine as a corepressor, that is made from methionine by the enzyme S-adenosylmethionine synthetase (EC 2.5.1.6) encoded by the essential gene metK. metK encoding homoserine transsuccinylase (EC 2.3.1.46), the first enzyme unique to the synthesis of methionine is another major control point for methionine production. Besides the transcriptional regulation of met A by MeU and MetR the enzyme is also feedback regulated by the end-products methionine and S-adenosylmethionine (Lee, L.-W et al.
  • the patent application JP2000139471 describes a process for the production of L-methionine using mutants in the genes metK and metK. In this case the precise location of the mutations has been determined.
  • the MetA mutant enzymes have partially lost the sensitivity to feed-back inhibition by methionine and S- adenosyl-methionine. Nevertheless their initial activities are decreased down to about 25% when compared to the wildtype enzyme, and at concentrations of 1 mM methionine for some mutants or 1 mM SAM for others another 25-90 % of the enzyme activity is lost.
  • the metK mutants were not characterized enzymatically but used in fermentations to increase the amount of methionine produced.
  • This invention relates to a method for the preparation of methionine, its precursors or products derived thereof in a fermentative process with a microorganism where L-homoserine is converted into O-succinyl-homoserine with a homoserine transsuccinylase, comprising the step of culturing the said microorganism on an appropriate medium and recovering methionine, its precursors or products derived thereof once produced, wherein the homoserine transsuccinylase is a mutated homoserine transsuccinylase with reduced sensitivity for the feedback inhibitors S- adenosylmethionine and methionine.
  • the invention also relates to the same method with microorganisms where the S-adenosylmethionine synthetase enzyme activity is reduced.
  • the present invention also concerns the mutated enzymes, nucleotide sequences coding for the said enzymes with reduced sensitivity or activity and microorganisms comprising the said nucleotide sequences as disclosed above and below.
  • the recombinant enzymes can be used together and can also be combined with several other changes in the corresponding microorganisms such as overexpression of genes or their deletion.
  • the gene encoding aspartokinase/homoserine dehydrogenase (meth or thrK) is over expressed and the gene encoding the methionine rcprcssor me is deleted.
  • genes and proteins are identified using the denominations of the corresponding genes in E. coli. However, and unless specified otherwise, use of these denominations has a more general meaning according to the invention and covers all the corresponding genes and proteins in other organisms, more particularly microorganisms.
  • PFAM protein families database of alignments and hidden Markov models; http://www.sanger.ac.uk/Software/Pfam ⁇ represents a large collection of protein sequence alignments.
  • Each PFAM makes il possible to visualize multiple alignments, sec protein domains, evaluate distribution among organisms, gain access to other databases, and visualize known protein structures.
  • COGs clusters of orthologous groups of proteins; http://www.ncbi.nlm nih. gov/COG/1 are obtained by comparing protein sequences from 43 fully sequcnccd genomes representing 30 major phylogenic lines. Each COG is defined from at least three lines, which permits the identification of former conserved domains.
  • the means of identifying homologous sequences and their percentage homologics arc well known to those skilled in the art, and include in particular the BLAST programs, which can be used from die website http://www.
  • ncbi.nlm.nih.gov BLAST/ wi th the default parameters indicated on that website.
  • the sequences obtained can then be exploited (e.g., aligned) using, for example, the programs CLUSTALW (http://www.ebi.ac.uk/clustal ⁇ v ⁇ or MULTALIN (http://prodcs.toulouse.inra.fr/multalin/cgi-bin/mul talin.pl). with the default parameters indicated on those websites.
  • CLUSTALW http://www.ebi.ac.uk/clustal ⁇ v ⁇
  • MULTALIN http://prodcs.toulouse.inra.fr/multalin/cgi-bin/mul talin.pl
  • «? to methionine and S-adcnosylmcthionine in comparison to the wild-type enzyme, according to the present invention, comprises at least one amino acid mutation when compared with the wild-type sequence.
  • the mutation is preferentially selected in the conserved regions coding for amino acids 24 to 30 or in the region coding for amino acids 58 to 65 or in the region coding for amino acids 292 to 298 with the first amino acid proline after the formylmethionine counting as number 1. All references to amino acid positions are made based on the homoserine succinyltransf erase encoded by the me i gene of E. coli represented in figure 2.
  • the protein sequence of the modified homoserine succinyltransferase according to the invention contains the amino acid mutation of at least one of the conserved regions sequences specified below.
  • at least one mutation is present in the conserved region 1 comprising the amino acid sequence defined below, in the N-terminal part of the wild type homoserine transsuccinylases, corresponding to amino acid 24 to 30 in the amino acid sequence of E. coli MetA shown in SEQ ID NO 1.
  • This non mutated conserved region 1 has the following sequence: X1-X2-X3-A-X4-X5-Q In which
  • XI represents E, D, T, S, L
  • T X2 represents D, S, K, Q, E, A, R
  • S X3 represents R, E, D, preferentially R
  • X4 represents Y, I, F, A, K, S, V, preferentially S X5 represents H, S, N, G, T, R, preferentially G.
  • the unmodified homoserine succinyltransferase conserved region 1 has the following amino acid sequence: T-S-R-A-S-G-Q.
  • at least one mutation is present in the conserved region 2, also in the N-terminal part of the wild type homoserine transsuccinylase, corresponding to amino acid 58 to 65 in the amino acid sequence of E. coli MetA shown in SEQ ID NO 1.
  • the non mutated conserved region 2 has the following formula: XI -X2-X3-P-L-Q-X4-X5 hi which
  • X 1 represents G, A, S, preferentially S X2 represents N, A, preferentially N X3 represents S, T, preferentially S X4 represents V, L, I, preferentially V
  • X5 represents N, E, H, D, preferentially D.
  • the unmodified homoserine succinyltransferase conserved region 2 has the following amino acid sequence: S-N-S-P-L-Q-V-D.
  • the homoserine succinyltransferase comprises at least one mutation in a conserved region in its C-terminal part, corresponding to amino acid 292 to 298 in the amino acid sequence of E. coli MetA shown in SEQ ID NO 1.
  • the non mutated conserved region 3 has the following formula: X1-Y-Q-X2-T-P-X3 In which XI represents V, I, M, preferentially V
  • X2 represents E, K, G, I, Q, T, S, preferentially
  • I X3 represents F, Y, preferentially Y.
  • the unmodified homoserine succinyltransferase conserved region 3 has the following amino acid sequence: V-Y-Q-I-T-P-Y.
  • the conserved alanine in conserved region 1 is replaced with another amino acid, more preferentially with a valine.
  • the modified conserved region has most preferentially the following amino acid sequence: T-S-R-V-S-G-Q.
  • the conserved amino acids L and/or Q in conserved region 2 are replaced with other amino acids.
  • leucine is replaced by phenylalanine and/or glutaminc is replaced with a glutamatc or aspartate.
  • the modified conserved region 2 has the following amino acid sequence: S-N-S-P-L-E-V-D.
  • the conserved amino acids L and or Q in conserved region 3 are replaced with another amino acid.
  • the metK gene is the homoserine transsuccinylase enzyme of E.
  • coli K12 represented by the SEQ ID NO 1 and sequences homologous to that sequence that have homoserine transsuccinylase activity and that share at least 80% homology, preferentially 90% homology with the amino acid sequence of SEQ ID NO 1.
  • Modified homoserine succinyltransferases may be obtained, for example, by selecting strains growing in the presence of methionine analogues such as ⁇ -methylmethionine, norleucine or ethionine. Preferentially these strains will be selected while growing in the presence of ⁇ - methylmethionine.
  • the present invention furthermore relates to nucleotide sequences, DNA or RNA sequences, which encode a mutated homoserine succinyltransferase according to the invention as defined above.
  • the DNA sequence is characterized by the fact that it comprises at least one mutation in the coding regions of the conserved regions 1 to 3 of the wild type met A gene, represented in SEQ ID NO 1 , the said mutation being not a silent mutation.
  • These DNA sequences can be prepared, for example, from the strains growing in the presence of methionine analogues.
  • the starting DNA fragment encompassing the modified met A gene is cloned in a vector using standard known techniques for preparing recombinant DNA.
  • DNA sequences can also be prepared, for example, by non-specific or by targeted mutagenesis methods from strains harboring the DNA sequence encoding wild-type homoserine succinyltransferase.
  • Non-specific mutations within the said DNA region may be produced, for example, by chemical agents (e.g. nitrosoguanidine, cthylmethancsulfonic acid and the like) and/or by physical methods and/or by PCR reactions, which are carried out under particular conditions. Methods for introducing mutations at specific positions within a DNA fragment are known and are described in Molecular Cloning: a Laboratory Manual, 1989,. 2 nd ed. Cold Spring Harbor Lab., Cold Spring Harbor, New York.
  • one or more mutations which cause the modified homoserine succinyltransferase to possess an amino acid sequence which leads to methionine and S-adenosylmethionine insensitivity can be introduced in the said DNA region of any met A gene.
  • one or more nucleotides in the DNA sequence encoding homoserine succinyltransferase are changed such that the amino acid sequence that is encoded by the gene exhibits at least one mutation.
  • Another method of producing feedback-resistant metK alleles consists in combining different point mutations which lead to feedback resistance, thereby giving rise to multiple mutants possessing new properties.
  • the modified S-adenosylmethioninc synthetase according to the invention has decreased activity in comparison to the wild-type enzyme, and has at least one mutation in its protein sequence when compared to the wild-type sequence.
  • the mutation is preferentially in one of the conserved regions defined below. All references to amino acid positions are made based on the S-adenosylmetiiionine synthetase encoded by the metK gene of E. coli.
  • the relative positions of corresponding conserved regions in other S-adenosylmethionine synthetases from different organisms can be found by a person skilled in the art by simple sequence alignment as represented in figure 4 with enzymes listed below:
  • Bordetella bronchiseptica >gi
  • the modified S-adenosylmethionine synthetase preferentially exhibits a specific activity which is at least five times less than that of the wild-type enzyme.
  • the protein sequence of a modified S-adenosylmethionine synthetase contains the amino acid mutation of at least one of the sequences specified below.
  • the amino acid changes concern the cystein at position
  • non mutated conserved region 1 comprising the amino acid sequence defined below:
  • XI represents I, V, L, T preferentially
  • I X2 represents T, K, S, R, preferentially T
  • X3 represents T, S, G, preferentially T
  • X4 represents S, N, T, K, E, R, P, N, A preferentially S.
  • the unmodified S-adenosylmethionine synthetase conserved region 1 has the following amino acid sequence: G-E-I-T-T-S.
  • at least one mutation is present in the conserved region
  • XI represents P, Q, A, S, preferentially P
  • X2 represents A, N, Q, S, F, preferentially N
  • X3 represents V, Q, Y, R, M, N preferentially Q
  • X4 represents K, D,N, T, S, A, E preferentially D.
  • the unmodified S-adenosylmethionine synthetase conserved region 2 has the following amino acid sequence: Q-S-P-D-I-N-Q-G-V-D.
  • at least one mutation is present in the conserved region 3, corresponding to amino acid 114 to 121 in the amino acid sequence of £ coli MetK shown in SEQ
  • the non mutated conserved region 3 comprising the amino acid sequence defined below: X1-G-A-G-D-Q-G-X2 wherein
  • XI represents Q, A, I, V, E, T, preferentially Q
  • X2 represents L, I, S, V, M, preferentially L.
  • the unmodified S-adcnosylmcthioninc synthetase conserved region 3 has the following amino acid sequence: Q-G-A-G-D-Q-G-L.
  • at least one mutation is present in the conserved region 4, corresponding to amino acid 137 to 144 in the amino acid sequence of E. coli MetK shown in SEQ
  • the non mutated conserved region 4 comprising the amino acid sequence defined below: X1-I-X2-X3-X4-H-X5-X6 wherein
  • XI represents S, P, T, A, preferentially P
  • X2 represents A, T, W, Y, F, S, N preferentially T
  • X3 represents M, Y, L, V preferentially Y X4 represents S, A, preferentially A
  • X5 represents K, R, E, D, preferentially R
  • X6 represents L, I, preferentially L.
  • the unmodified S-adenosylmethionine synthetase conserved region 4 has the following amino acid sequence: P-I-T-Y-A-H-R-L.
  • at least one mutation is present in the conserved region 5, corresponding to amino acid 159 to 163 in the amino acid sequence of E. coli MetK shown in SEQ
  • X1-L-X2-X3-D wherein XI represents W, F, Y, V, E preferentially W
  • X2 represents R, G, L, K, preferentially R
  • X3 represents P, L, H, V, preferentially P.
  • the unmodified S-adenosylmethionine synthetase conserved region 5 has the following amino acid sequence: W-L-R-P-D.
  • Preferentially at least one mutation is present in the conserved region 6, corresponding to to amino acid 183 to 189 in the amino acid sequence of E. coli MetK shown in SEQ ID NO 2, the non mutated conserved region 6 comprising the amino acid sequence defined below.
  • X2 represents V, L, I, preferentially V
  • X3 represents V, L, I, M, preferentially L
  • X4 represents T, V, A, S, H, preferentially T.
  • the unmodified S-adenosylmethionine synthetase conserved region 6 has the following amino acid sequence: V-V-L-S-T-Q-H.
  • the mutations are introduced in the conserved region 7, corresponding to amino acid 224 to 230 in the amino acid sequence off.
  • coli MetK shown in SEQ ID NO 2, the non mutated conserved region 7 comprising the amino acid sequence defined below: X1-N-P-X2-G-X3-F In which
  • the unmodified S-adenosylmethionine synthetase conserved region 7 has the following amino acid sequence: I-N-P-T-G-R-F.
  • the mutations are introduced into the conserved region 8, corresponding to amino acid 231 to 237 in tire amino acid sequence ofE. coli MetK shown in SEQ ID NO 2, the non mutated conserved region 8 comprising the amino acid sequence defined below: X1-X2-G-X3-P-X4-X5 In which
  • XI represents T, V, 1, Y, E, preferentially V X2 represents V, I, L, N, preferentially I X3 represents G, S, preferentially G
  • the unmodified S-adenosylmethionine synthetase conserved region 8 has the following amino acid sequence: V-l-G-G-P-M-G.
  • the mutations arc introduced in the conserved region 9, corresponding to amino acid 246 to 253 in the amino acid sequence of E. coli MetK shown in SEQ ID NO 2, the non mutated conserved region 9 comprising the amino acid sequence defined below: X1-X2-D-T-Y-G-G In which XI represents M, I, preferentially I X2 represents V, I preferentially I.
  • the unmodified S-adenosylmethionine synthetase conserved region 9 has the following amino acid sequence: I-V-D-T-Y-G-G.
  • the mutations are introduced into the conserved region 10, corresponding to amino acid 269 to 275 in the amino acid sequence off.
  • coli MetK shown in SEQ ID NO 2 the non mutated conserved region 10 comprising the amino acid sequence defined below: K-V-D-R-S-X1-X2 In which XI represents A, G, preferentially A X2 represents A, S, L, preferentially A.
  • the unmodified S-adenosylmethionine synthetase conserved region 10 has the following amino acid sequence: K-V-D-R-S-A-A
  • at least one mutation is present in the conserved region 11.
  • the unmodified S-adcnosylmethioninc synthetase harbors the conserved region in its C-terminal part with the following amino acid sequence: XI -X2-Q-X3-X4-Y-A-I-G-X5-X6 In which
  • XI represents L, E, I, Q, T preferentially
  • E X2 represents V
  • I L
  • I X3 represents V
  • 1 preferentially V X4 represents A
  • S preferentially S X5 represents V
  • a preferentially V X6 represents A, V, T, S, preferentially A.
  • This region corresponds to amino acid 295 to 305 in the amino acid sequence of E. coli
  • the unmodified S-adenosylmethionine synthetase conserved region 11 has the following amino acid sequence: E-I-Q-V-S-Y-A-I-G-V-A.
  • the mutations are introduced into the N- terminus of the protein leading to a frameshift and changes in the last 6 amino acids.
  • the serine in conserved region 1 is replaced with another amino acid.
  • the serine is replaced with a asparagine.
  • the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 1: G-E-I-T-T-N.
  • the conserved glycine in conserved region 2 is replaced with another amino acid.
  • the conserved glycine is replaced with a serine.
  • the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 2: Q-S-P-D-I-N-Q-S-V-D.
  • the conserved glycine in conserved region 3 is replaced with another amino acid.
  • the conserved glycine is replaced with a serine.
  • the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 3: Q-S-A-G-D-Q-G-L.
  • the S-adenosyhnethionine synthetase with decreased activity preferentially die conserved histidine and/or the semi-conserved proline in conserved region 4 is/are replaced with other amino acids.
  • the conserved histidine is replaced with a lyrosine and/or the semi -conserved proline is replaced with a leucine.
  • the modified S-adenosylmethionine synthetase has one of the following amino acid sequences in conserved region 4: P-I-T-Y-A-Y-R-L, L-I-T-Y-A-H-R-L, L-I-T-Y-A-Y-R-L.
  • the semi- conserved arginine in conserved region 5 is replaced with another amino acid.
  • the arginine is replaced with a cysteine.
  • the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 5: W-L-C-P-D.
  • the conserved histidine or semi-conserved valine in conserved region 6 is replaced with another amino acid.
  • the conserved histidine is replaced with a tyrosine and/or the valine with aspartate.
  • the modified S-adenosylmethionine synthetase has one of the three following amino acid sequences in conserved region 6: V-V-L-S-T-Q-Y V-D-L-S-T-Q-H V-D-L-S-T-Q-Y.
  • the semi- conserved threonine in conserved region 7 is replaced with another amino acid.
  • the threonine is replaced with an isoleucine.
  • the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 7: 1-N-P-I-G-R-F.
  • the conserved proline in conserved region 8 is replaced with another amino acid.
  • the proline is replaced with a serine.
  • the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 8: V-I-G-G-S-M-G.
  • the second conserved glycine in conserved region 9 is replaced with another amino acid.
  • the glycine is replaced with an aspartate.
  • the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 9: 1-V-D-T-Y-G-D.
  • the conserved serine in conserved region 10 is replaced with another amino acid.
  • the serine is replaced with a phenylalanine.
  • the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 10: K-V-D-R-F-A-A.
  • the conserved isoleucine in conserved region 1 1 is replaced with other amino acids.
  • isoleucine is replaced by leucine.
  • the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 11: E-I-Q-V-S-Y-A-L-G-V-A.
  • the present invention furthermore relates to nucleotide sequences, DNA or RNA sequences, which encode a mutated S-adenosylmethionine synthetase according to the invention as defined above.
  • these DNA sequences are characterized by the fact that they comprise al least one mutation in the coding DNA sequence regions for the conserved regions 1 to 11 of the wild type metK gene, represented as wild type in SEQ ID NO 2, the said mutation being not a silent mutation.
  • the metK gene is the S-adenosylmethionine synthetase off.
  • coli K12 represented by the SEQ ID NO 2 and sequences homologous to that sequence that have S- adenosylmethionine synthetase activity and that share at least 80% homology, preferentially 90% homology with the amino acid sequence of SEQ ID NO 2.
  • the mutated S-adenosylmethionine synthetase genes described above may be obtained by conventional techniques known to the person skilled in the art disclosed above and below, including random or targeted mutagencsis or synthetic DNA construction.
  • the metK gene encoding modified homoserine succinyltransferase and/or the metK gene encoding modified S-adenosylmethionie synthetase may be encoded chromosomally or extrachromosomally. Chromosomally there may be one or several copies on the genome that can be introduced by methods of recombination known to the expert in the field. Extrachromosomally both genes may be carried by different types of plasmids that differ with respect to their origin of replication and thus their copy number in the cell.
  • the metK and/or metK gene may be expressed using promoters with different strength that need or need not to be induced by inducer molecules. Examples are the promoter Ptrc, Ptac, Plac, the lambda promoter cl or other promoters known to the expert in the field.
  • MetA and/or MetK expression may be boosted or reduced by elements stabilizing or destabilizing the corresponding messenger RNA (Carrier and Keasling (1998) Biotechnol. Prog. 15, 58-64) or the protein (e.g. GST tags, Amersham Biosciences).
  • the present invention also relates to microorganisms which contain a feedback-resistant metK allele according to the invention and eventually a metK allele with reduced activity according to the invention.
  • Such strains are characterized by the fact that they possess a methionine metabolism which is deregulated by at least one feedback-resistant metK allele and/or by a reduced production of SAM caused by a MetK enzyme with reduced activity.
  • Novel strains may be prepared from any microorganism in which methionine metabolism proceeds by the same metabolic pathway.
  • Gram-negative bacteria in particular E. coli, are especially suitable.
  • the modified homoserine succinyltransferase enzyme and S- adcnosylmcthioninc synthetase are transformed in a host strain using customary methods.
  • the screening for strains possessing modified homoserine succinyltransferase and S-adenosylmethionine synthetase properties is, for example, realized using enzymatic tests.
  • the homoserine succinyltransferase activity can be determined in an enzymatic test with homoserine and succinyl-CoA as substrates.
  • the reaction is started by adding the protein extract containing the homoserine succinyltransferase enzyme, and the formation of O- succinylhomoserine is monitored by GC-MS after protein precipitation and derivatization with a silylating reagent. Feedback inhibition is tested in the presence of methionine and S- adenosylmethionine in the reaction mixture.
  • S-adenosylmethionine synthetase activity can be determined in an enzymatic test with methionine and ATP as substrates.
  • the reaction is started by adding the protein extract containing the S-adenosylmethionine synthetase enzyme, and the formation of S-adenosylmethionine is monitored by FIA-MS/MS.
  • FIA-MS/MS FIA-MS/MS.
  • E. coli strains in which the endogenous met A and metK genes are inactivated and complemented by novel recombinant genes.
  • the feedback-resistant metK alleles and the metK alleles with reduced activity render it possible to abolish the control at important biosynthetic control points, thereby amplifying the production of a large number of compounds which are situated downstream of aspartate.
  • the invention relates to the preparation of L-methionine, its precursors or compounds derived thereof, by means of cultivating novel microorganisms.
  • the above-described products are classified below as compound (I).
  • An increase in the production of compound (I) can be achieved by changing the expression levels or deleting the following genes implicated in the production of aspartate, a precursor of compound (I).
  • Rhizobium etli accession nombct r* 1 39"> tra> be introduced ge etic engineering wi I cob and An additional JKC tease m tac psodnet ⁇ n of compound (!) can be achieved oics vpressm" ⁇ >e ⁇ es of the h sme/mcthionine paihvtsv.
  • a further increase in the production of (I) is possible by means of deleting the gene for the repressor protein MetJ, responsible for the down-regulation of the methionine regulon as was suggested in JP 2000157267-A 3 (see also GenBank gl790373).
  • Production of (I) may be further increased by using an altered metB allele that uses preferentially or exclusively H 2 S and thus produces homocysteine from O succinylhomoserine as has been described in the patent application PCT N° PCT/FR04/00354, which content is incorporated herein by reference.
  • the gene encoding aspartokinase/homoserine dehydrogenase is over expressed and/or die gene encoding methionine repressor metJ is deleted in the microorganism of the present invention and in the microorganism used in the method according to the invention.
  • the aspartokinase/homoserine dehydrogenase is encoded by a feed-back deregulated ThrA allele.
  • feed-back deregulation may be obtained by introducing the mutation Phe318Ser in the ThrA enzyme. Enzyme position is given here by reference to the ThrA sequence disclosed in gencbank accession number V00361.1 (gl786183).
  • the metK and metK alleles described above may be used in eukaryotes or prokaryotes.
  • the organism used is a prokaryote.
  • the organism is either E. coli or C. glutamicum.
  • the invention also concerns the process for the production of compound (I).
  • Compound (I) is usually prepared by fermentation of the designed bacterial strain.
  • the terms 'culture' and 'fermentation' are used indifferently to denote the growth of a microorganism on an appropriate culture medium containing a simple carbon source.
  • a simple carbon source is a source of carbon that can be used by those skilled in the art to obtain normal growth of a microorganism, in particular of a bacterium.
  • the microorganisms can be an assimilable sugar such as glucose, galactose, sucrose, lactose or molasses, or by-products of these sugars.
  • An especially preferred simple carbon source is glucose.
  • Another preferred simple carbon source is sucrose.
  • ⁇ ⁇ • ' Those skilled in the art are able to define the culture conditions for the microorganisms according to the invention.
  • the bacteria are fermented at a temperature between 20°C and 55°C, preferentially between 25°C and 40°C, and more specifically about 30°C for C. glutamicum and about 37°C for E. coli.
  • the fermentation is generally conducted in fermenters with an inorganic culture medium of known defined composition adapted to the bacteria used, containing at least one simple carbon source, and if necessary a co-substrate necessary for the production of the metabolite.
  • the inorganic culture medium for E. coli can be of identical or similar composition to an M9 medium (Anderson, 1946, Proc. Natl. Acad. Sci. USA 32:120-128), an M63 medium (Miller, 1992; A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) or a medium such as defined by Schaefer et al. (1999, Anal. Biochem.
  • the inorganic culture medium for C. glutamicum can be of identical or similar composition to BMCG medium (Liebl et al., 1989, Appl. Microbiol. Biotechnol. 32: 205- 210) or to a medium such as that described by Riedel et al. (2001, J. Mol. Microbiol. Biotechnol. 3: 573-583).
  • the media can be supplemented to compensate for auxotrophies introduced by mutations.
  • fermentation compound (I) is recovered and purified if necessary.
  • the methods for the recovery and purification of compound (I) such as methionine in the culture media are well known to those skilled in the art.
  • Figure 1A/B Methionine metabolism in Escherichia coli.
  • Figure 2 Alignment of wildtype and recombinant metK genes obtained upon selection on ⁇ - methyl-methionine. conserveed residues are represented by light grey boxes and mutated residues are indicated by white boxes.
  • - Figure 3 Alignment of MetA sequences from different microorganisms.
  • Figure 4 Alignment of MetK sequences from different microorganisms.
  • Figure 5 Amino acid replacements in the E. coli MetK protein. Amino acids above the coherent lines indicate the position and the replacing amino acid.
  • Isolation of E. coli mutants containing homoserine transsuccinylase enzymes which show decreased feedback-sensitivity towards methionine and S-adenosylmethionine Isolation ofE. coli strains growing on a-methylmethionine ⁇ -mcthylmethionine is a growth-inhibitory analogue of methionine producing an immediate effect on the growth rate of E. coli at very low concentrations (minimal inhibitory concentration of l ⁇ g/ml and minimal concentration for a maximal inhibition 5 ⁇ g/ml, Rowbury et al., 1968). Analogues can mimic methionine in the feedback inhibition of homoserine transsuccinylase and interfere with protein synthesis without being metabolized.
  • the medium used for the ⁇ -methyl-methionine analogue resistant was a minimal medium containing (per liter): K 2 HP0 4 8g, Na 2 HP0 4 2g, (NH 4 ) 2 S0 4 0.75g, (NH 4 ) 2 HP0 4 8g, NH 4 C1 0.13g, citric acid 6.55g, MgS0 4 2.05g, CaCl 2 40mg, FeS0 4 40mg, MnS0 20mg, CoCl 2 8 mg, ZnS0 4 4mg, (NH 4 ) 2 Mo 2 0 7 2.8mg, CuCl 2 2mg, H 3 BO 3 lmg.
  • met A gene was PCR-amplified using Taq polymerase and the following primers: MetAF (SEQ ID NO 3): tcaccttcaacatgcaggctcgacattggc (421 1759-421 1788) MetAR (SEQ ID NO 4): ataaaaaaggcacccgaaggtgcctgaggt (4212857-4212828).
  • MetAF SEQ ID NO 3
  • MetAR SEQ ID NO 421 1759-421 1788
  • MetAR SEQ ID NO 4212857-4212828.
  • metKl 1 CAG was exchanged for a GAG leading to the replacement of Q by E.
  • metK* 13 TTG was exchanged for a TTT leading to the replacement of L by F.
  • metK* 14 GCG was exchanged for GTG leading to the replacement of A by V (see fig.2).
  • succinyl-CoA 4 mM succinyl-CoA. Methionine and/or S-adenosylmethionine were added as indicated.
  • the succinylhomoserine produced by homoserine transsuccinylase enzymes was quantified by GC-MS after dcrivatization with tcrt-butyldimcthylsilyltrifluoroacetamide (TBDMSTFA).
  • TBDMSTFA tcrt-butyldimcthylsilyltrifluoroacetamide
  • the mutated homoserine transsuccinylase enzymes thus show decreased feedback- sensitivity towards methionine and S-adenosylmethionine.
  • Isolation of E. coli mutants containing S-adcnosylmethionine synthetase enzymes with reduced activity Isolation ofE. coli strains growing on norleucine Norleucine is a growth-inhibitory analogue of methionine. At higher concentrations (50 mg/1) only mutant strains are able to grow in a medium containing the analogue. Most of these mutations map to the metK and met] loci. E. coli was routinely grown aerobically at 37°C in LB supplemented when needed with the appropriate antibiotic.
  • the medium used for the norleucine analogue resistant was a minimal medium containing (per liter): K 2 HP0 4 8g, Na 2 HP0 4 2g, (NH 4 ) 2 S0 0.75g, (NH 4 ) 2 HP0 4 8g, NH 4 C1 0.13g, citric acid 6.55g, MgSQ, 2.05g, CaCl 2 40mg ; sFeSO 4 40mg, MnS0 4 20mg, CoCl 2 8 mg, ZnS0 4 4mg, (NH 4 ) 2 Mo 2 0 7 2.8mg, CuCl 2 2mg, H 3 B0 3 lmg.
  • the pH was adjusted to 6.7 and the medium sterilized. Before use, glucose 1 g/l and thiamine 1 Omg/1 were added. 1*10 8 cells / ml from an overnight culture in minimal medium of the wild type strain
  • Gcnomic DNA was prepared from cultures grown in LB liquid medium. The cell pellet was washed once with sterile water and resuspended in 30 ⁇ l of sterile water. Cells were broken by heating 5 minutes at 95°C and the debris was extracted.
  • DNA was PCR-amplified with Taq polymerase using the following primers: MetKpF : cccggctggaagtggcaacacg (3084372-3084393) (SEQ ID NO 05) MetKR : gccggatgcggcgtgaacgcctatcc (3085956-3085931) (SEQ ID NO 06).
  • the metK gene was sequenced. In 10 clones point mutations were detected which led to amino acid substitutions. In clone efK*59/105 AGC was exchanged for AAC leading to the replacement of S by N (position 59) and GGC was exchanged for AGC leading to the replacement of G by S (position 105).
  • Table 2 S-adenosylmethionine synthetase activities (MAT) of WT and MetK* mutants.
  • MAT S-adenosylmethionine synthetase activities
  • E. coli strains for the production of O-succinylhomoserine or methionine by overexpressing altered homoserine transsuccinylase and other enzymes of the methionine biosynthesis pathway For the construction of E. coli strains that allow the production of O-succinylhomoserinc, a plasmid overexpressing the metL gene, coding for homoserine dehydrogenase and aspartokinase was introduced into strains harboring different alleles of met A.
  • the plasmid overexpressing metL was constructed as follows: The following two oligonucleotides were used: - MctLF with 32 bases (SEQ ID NO 7): TATTCatgagtgtgattgcgcaggcaggggcg with
  • HDH Homoserine dehydrogenase II
  • MG1655 pKD46
  • McUR and MetTF defined below.
  • the strain retained is designated MG1655 ( ⁇ metf ::Cm) metK*.
  • MetJR (SEQ ID NO 11): ggtacagaaaccagcaggctgaggatcagc (homologous to the sequence from 4125431 to 4125460).
  • MetBR (SEQ ID NO 12): ttcgtcgtcatttaacccgctacgcactgc (homologous to the sequence from 4126305 to 4126276).
  • the chloramphenicol resistance cassette was then eliminated.
  • the plasmid pCP20 carrying recombinase FLP acting at the FRT sites of the chloramphenicol resistance cassette was introduced into the recombinant strains by electroporation.
  • homoserine succinyltransferase alleles expressing enzymes with further reduced feed-back sensitivity methionine and SAM other metK mutants were constructed by site directed mutagenesis. Initially the metK and met A*l 1 allele were cloned into the vector pTRC99A (Stratagene).
  • Ncol and £ ⁇ r ⁇ RI were introduced at the N- and C -terminus of die metK gene, respectively.
  • the resulting PCR fragments were restricted by Ncol and EcoRl and cloned into the vector pTRC99A (Stratagene) previously cut by Ncol and EcoRl. Plasmid preparations were examined for the presence of inserts of die correct size and the DNA sequences of metK and met A*l I were verified by sequencing.
  • MetAQ64DF (EcoRV) (SEQ ID NO 19): Gctttcaaactcacctttggatgtcgatatccagctgttgc MetAQ64DR (EcoRV) (SEQ ID NO 20) gcaacagctggatatcgacatccaaaggtgagtttgaagc and pTRC/nef A as matrix.
  • pTRCmetA*17 A27V + Q64D
  • the following oligonucleotides were used: MetAA28VF (Xbal) (SEQ ID NO 17) and MetAA28VR (Xbal) (SEQ ID NO 18); and pTRC efA*16 as matrix.
  • Plasmids were verified by sequencing. To transfer the alleles metK*15, metK*l6 and metK*17 onto the metK locus on the chromosome, a metK deletion was constructed in the AmetJ background applying the same strategy used for the met] deletion.
  • DmetAF (4211866-4211945)
  • SEQ ID NO 21 ttcgtgtgccggacgagctacccgccgtcaatttcttgcgtgaagaaaacgtctttgtgatgacaacttctcgtgcgtctTGTAGGCTGG AGCTGCTTCG DmetAR (4212785-4212706)
  • SEQ ID NO 22 AtccagcgttggattcatgtgccgtagatcgtatggcgtgatctggtagacgtaatagttgagccagttggtaacagtaCATATGAAT ATCCTCCTTAG
  • the region indicated in lower case corresponds to the sequence between metK and yjaB.
  • the region in upper case is used to amplify the kanamycin resistance cassette (Datsenko & Wanner, 2000).
  • Numbers in parentheses correspond to the reference sequence on the website http://gcnolist.pastcur.fr/Colibri .
  • the resulting deletion was verified using the following oligonucleotides.
  • Numbers in parentheses correspond to Uic reference sequence on the website http:// ⁇ cnolist.pasteur fr/Colibri .
  • MetAF SEQ ID NO 3
  • MetAR SEQ ID NO 4
  • the plasmid pKD46 was introduced into the strain DmetJ OmelK (Datsenko and Wanner, 2000).
  • the alleles were amplified from the vectors pTROwe/A*15, pTRCmetK*16 and pTRC/wefA* 17 using the following oligonucleotides: MetArcF (4211786-4211884) (SEQ ID NO 23) Ggcaaattttctggttatcttcagctatctggatgtctaaacgtataagcgtatgtagtgaggtaatcaggttatgccgattcgtgtgccggacga gc MetArcR (4212862-4212764) (SEQ ID NO 24) Cggaaataaaaaggcacccgaaggtgcctgaggtaaggtgctgaatcgcttaacgatcgact
  • the resulting strains were cultivated in minimal medium, crude extracts were prepared and the activity of MetA was determined.
  • the newly constructed alleles are less sensitive to the inhibition by methionine and SAM than the alleles described above.
  • the allele metA* 15 with high intrinsic activity uiat cannot be inhibited by SAM and methionine.
  • E. coli strains for the production of O-succinyl homoserine or methionine by combining feed-back resistant MetA alleles with MetK alleles with decreased activity
  • a kanamycin resistance cassette was introduced between die metK and gal? gene.
  • the homologous recombination strategy described by Datsenko & Wanner (2000) is used. This strategy allows the insertion of a kanamycin resistance cassette, while deleting most of the gene concerned.
  • oligonucleotides are used: - DMetKFscr with 100 bases (SEQ ID NO 25): ccgcccgcacaataacatcattcttcctgatcacgtttcaccgcagattatcatcacaactgaaaccgattacaccaaccTGTAGGCTGG AGCTGCTTCG with - a region (lower case) homologous to the sequence of the region between metK and gal? (sequence 3085964 to 3086043, reference sequence on the website http://genolist.pasteur.fr/Colibri/).
  • the oligonucleotides DMetKFscr and DmetKRscr are used to amplify the kanamycin resistance cassette from the plasmid pKD4.
  • the PCR product obtained is then introduced by electroporation into the strain MG1655 (pKD46) in which the expressed Red recombinase enzyme allows the homologous recombination.
  • the kanamycin resistant transfomiants arc then selected and the insertion of the resistance cassette is verified by a PCR analysis with the oligonucleotides MetKFscr and MetKRscr defined below.
  • the strain retained is designated MG1655 (metK, Km).
  • MetKFscr SEQ ID NO 27: gcgcccatacggtctgatlcagatgctgg (homologous to the sequence, from 3085732 to 3085760).
  • MetKRscr SEQ ID NO 28: gcgccagcaatlacaccgatatccaggcc (homologous to the sequence from 3086418 to 3086390).
  • the protocol followed is implemented in 2 steps with the preparation of the phage l sale of the strain MG1655 (melK, Km) and then transduction into strain MG1655 AmetJ metK*l l pTRCmelL.
  • the construction of the strain ⁇ metK, Km) is described above.
  • phage lvsate PI Inoculation with 100 ⁇ l of an overnight culture of the strain MG1 55 (melK, Km) of 10 ml of LB + Km 50 ⁇ g/ml + glucose 0.2 % + CaCl 2 5 mM Incubation for 30 min at 37°C wiUi shaking - Addition of 100 ⁇ l of phage lysale PI prepared on d e wild strain MG1655 (about 1.10 9 phage/ml) Shaking at 37°C for 3 hours until all the cells were lysed Addition of 200 ⁇ l chloroform and vortexing Centrifugation for 10 min at 4500 g to eliminate cell debris Transfer of supernatant to a sterile tube and addition of 200 ⁇ l chloroform Storage of lysate at 4°C.
  • the kanamycin resistant transfomiants are then selected and the insertion of the region containing (metK, Km) is verified by a PCR analysis with the oligonucleotides MetKFscr and MetKRscr.
  • the strain retained is designated MG1655 AmetJ metK*l l pTRCmefL metK*.
  • the kanamycin resistance cassette can then be eliminated.
  • FLP recombinase acting at the FRT sites of the kanamycin resistance cassette is then introduced into the recombinant sites by electroporation. After a series of cultures at 42°C, the loss of the kanamycin resistance cassette is verified by a PCR analysis with the same oligonucleotides as used • previously (MetKFscr and MetKRscr).
  • Fermentation of E. coli production strains and analysis of yield Production strains were initially analyzed in small Erlenmeyer flask cultures using modified M9 medium (Anderson, 1946, Proc. Natl. Acad. Sci. USA 32:120-128) that was supplemented with 5 g/1 MOPS and 5 g/1 glucose. Carbcnicillin was added if necessary at a concentration of 100 mg/1. An overnight culture was used to inoculate a 30 ml culture to an OD600 of 0.2. After the culture had reached an OD600 of 4.5 to 5, 1 ,25 ml of a 50% glucose solution and 0.75 ml of a 2M MOPS (pH 6.9) were added and culture was agitated for 1 hour.
  • a thrA* allele with reduced feed-back resistance to threonine is expressed from the plasmid pCL1920 (Lerner & 10 Inouye, 1990, NAR 18, 15 p 4631) using the promoter Ptrc.
  • plasmid pME107 thrK was PCR amplified from genomic DNA using the following oligonucleotides: Bsp lt rA (SEQ ID NO 29): ttaTCATGAgagtgttgaagttcggcggtacatcagtggc Sfot ⁇ lthrA (SEQ ID NO 30): ttaCCCGGGccgcccccgagcacatcaaacccgacgc
  • the PCR amplified fragment is cut with the restriction enzymes BspHl and Smal and 15 cloned into the Ncol / Smal sites of the vector pTRC99A (Stratagene).
  • the plasmid pMElOl is constructed as follows.
  • the plasmid pCL1920 is PCR amplified using the oligonucleotides PME101F and PME101R and the BstZlT -Xmn fragment from the vector pTRC99A harboring the lacl gene and the Ptrc promoter is inserted into the amplified vector.
  • the resulting vector and the vector harboring the thrK gene are restricted by Apal 0 and Smal and the thrK containing fragment is cloned into the vector pMElOl.
  • F318S is introduced by site-directed mutagenesis (Stratagene) using the oligonucleotides ThrAF F318S for and ThrAR F318S, resulting in the vector pME 107.
  • the vector pME 107 was introduced into the AmetJ metK* 11 strains with differing metK* alleles.
  • PME101F (SEQ ID NO 31): Ccgacagtaagacgggtaagcctg PME101R (SEQ ID NO 32): Agcttagtaaagccctcgctag ThrAF F318S (Smal) (SEQ ID NO 33): Ccaatctgaataacatggcaatgtccagcgtttctggccggg ThrAR F318S (Smal) (SEQ ID NO 34): Cccgggccagaaacgctggacattgccatgttattcagattgg Strains that produced substantial amounts of metabolites of interest were subsequently tested under production conditions in 300 ml fermentors (DASG1P) using a fed batch protocol.
  • DSG1P 300 ml fermentors
  • the fermentor was filled with 145 ml of modified minimal medium and inoculated with 5 ml of preculture to an optical density (OD600nm) between 0.5 and 1.2.
  • the temperature of the culture was maintained constant at 37 °C and the pH was permanently adjusted to values between 6.5 and 8 using an NH OH solution.
  • the agitation rate was maintained between 200 and 300 rpm during the batch phase and was increased to up to 1000 rpm at the end of the fed-batch phase.
  • the concentration of dissolved oxygen was maintained at values between 30 and 40% saturation by using a gas controller.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne l'utilisation d'enzymes recombinantes homosérine transsuccinylase présentant une sensibilité modifiée à la réaction (MetA*) ainsi que des enzymes recombinantes S-adénosyl méthionine synthétase présentant une activité réduite (MetK*) pour la production de méthionine, de ses précurseurs ou de ses dérivés.
PCT/EP2005/052180 2004-05-12 2005-05-12 Enzyme recombinante presentant une sensibilite modifiee a la reaction WO2005108561A2 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
PL05742717T PL1747269T3 (pl) 2004-05-12 2005-05-12 Rekombinowany enzym o zmienionej wrażliwości na sprzężenie zwrotne
JP2007512216A JP5164566B2 (ja) 2004-05-12 2005-05-12 フィードバック感受性が変化した組み換え酵素
CN2005800153152A CN1954067B (zh) 2004-05-12 2005-05-12 改变反馈敏感度的重组酶
US11/579,907 US8795990B2 (en) 2004-05-12 2005-05-12 Recombinant enzyme with altered feedback sensitivity
BRPI0510819-5A BRPI0510819B1 (pt) 2004-05-12 2005-05-12 método para a preparação de metionina, seus precursores ou produtos derivados da mesma e microorganismo recombinante
MXPA06013125A MXPA06013125A (es) 2004-05-12 2005-05-12 Enzima recombinante con sensibilidad alterada a la retroalimentacion.
ES05742717.1T ES2535816T3 (es) 2004-05-12 2005-05-12 Enzima recombinante con sensibilidad alterada a la retroalimentación
EP05742717.1A EP1747269B1 (fr) 2004-05-12 2005-05-12 Enzyme recombinante presentant une sensibilite modifiee a la reaction

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IBPCT/IB2004/001901 2004-05-12
PCT/IB2004/001901 WO2005111202A1 (fr) 2004-05-12 2004-05-12 Enzyme recombinee avec sensibilite de retroaction modifiee

Publications (2)

Publication Number Publication Date
WO2005108561A2 true WO2005108561A2 (fr) 2005-11-17
WO2005108561A3 WO2005108561A3 (fr) 2006-07-20

Family

ID=34957660

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/IB2004/001901 WO2005111202A1 (fr) 2004-05-12 2004-05-12 Enzyme recombinee avec sensibilite de retroaction modifiee
PCT/EP2005/052180 WO2005108561A2 (fr) 2004-05-12 2005-05-12 Enzyme recombinante presentant une sensibilite modifiee a la reaction

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/IB2004/001901 WO2005111202A1 (fr) 2004-05-12 2004-05-12 Enzyme recombinee avec sensibilite de retroaction modifiee

Country Status (8)

Country Link
US (1) US8795990B2 (fr)
EP (1) EP1747269B1 (fr)
JP (1) JP5164566B2 (fr)
CN (1) CN1954067B (fr)
ES (1) ES2535816T3 (fr)
MX (1) MXPA06013125A (fr)
PL (1) PL1747269T3 (fr)
WO (2) WO2005111202A1 (fr)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008013432A1 (fr) 2006-07-28 2008-01-31 Cj Cheiljedang Corporation Micro-organisme produisant un précurseur de l-méthionine et procédé de production de l-méthionine et d'acide organique à partir du précurseur de l-méthionine
WO2008034338A1 (fr) * 2006-08-31 2008-03-27 Bioright Worldwide Company Limited Mutants de la s-adénosylméthionine synthétase, les adn les codant et les utilisations de ces mutants
WO2008127240A1 (fr) * 2007-04-11 2008-10-23 Cargill, Incorporated Compositions et méthodes de production de méthionine
EP2108693A2 (fr) 2008-04-04 2009-10-14 CJ Cheiljedang Corporation Micro-organisme produisant un précurseur de méthionine L et procédé de production du précurseur de méthionine L utilisant le microorganisme
JP2009247354A (ja) * 2008-04-04 2009-10-29 Cj Cheiljedang Corp Ｌ−メチオニン前駆体生産菌株およびこれを用いたｌ−メチオニン前駆体の生産方法
CN102002472A (zh) * 2009-08-28 2011-04-06 Cj第一制糖株式会社 产生o-乙酰高丝氨酸的微生物和利用所述微生物产生o-乙酰高丝氨酸的方法
CN102002473A (zh) * 2009-08-28 2011-04-06 Cj第一制糖株式会社 产生o-乙酰高丝氨酸的微生物和利用所述微生物产生o-乙酰高丝氨酸的方法
WO2011073122A1 (fr) 2009-12-14 2011-06-23 Metabolic Explorer Utilisation de promoteurs inductibles dans la production de méthionine
WO2011080542A1 (fr) 2009-12-30 2011-07-07 Metabolic Explorer Augmentation de la production de méthionine par surexpression de succinate déshydrogénase
US8017362B2 (en) 1998-11-17 2011-09-13 Ajinomoto Co., Inc. Method for producing L-methionine by fermentation
EP2314710A3 (fr) * 2006-01-04 2012-02-22 Metabolic Explorer Procédé de production de méthionine par la culture de micro-organismes modifiés pour améliorer la production de cystéine
WO2012087039A2 (fr) 2010-12-21 2012-06-28 씨제이제일제당 (주) Polypeptide de variant possédant une activité d'homosérine acétyltransférase et microorganisme l'exprimant
CN102703398A (zh) * 2007-04-11 2012-10-03 Cj第一制糖株式会社 组合物及甲硫氨酸的生产方法
WO2012137689A1 (fr) 2011-04-01 2012-10-11 味の素株式会社 Procédé pour la production de l-cystéine
EP2573189A1 (fr) 2007-10-02 2013-03-27 Metabolic Explorer Augmentation du rendement de la méthionine
CN103710320A (zh) * 2007-04-11 2014-04-09 Cj第一制糖株式会社 组合物及甲硫氨酸的生产方法
US9234223B2 (en) 2011-04-01 2016-01-12 Ajinomoto Co., Inc. Method for producing L-cysteine
US9850514B2 (en) 2012-09-19 2017-12-26 Genentech, Inc. Methods and compositions for preventing norleucine misincorporation into proteins
WO2018007565A1 (fr) 2016-07-08 2018-01-11 Metabolic Explorer Procédé de production fermentative de méthionine ou de sa forme analogue hydroxy par des micro-organismes comprenant des gènes codant un système de phosphotransférase de sucre (pts)
EP3296404A1 (fr) 2016-09-15 2018-03-21 Evonik Degussa GmbH Micro-organisme modifie pour production de methionine

Families Citing this family (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006082252A2 (fr) * 2005-02-07 2006-08-10 Metabolic Explorer Procede destine a la production enzymatique d'alpha-cetobutyrate
CA2687559A1 (fr) * 2007-06-25 2009-04-02 Plant Bioscience Limited Enzymes mises en jeu dans la synthese de triterpenes
WO2010022763A1 (fr) * 2008-08-25 2010-03-04 Metabolic Explorer Procédé de préparation de 2-hydroxy-isobutyrate
CN102341502B (zh) 2008-12-31 2015-07-01 代谢探索者公司 用于制备二醇的方法
FR2951195B1 (fr) 2009-10-14 2014-01-31 Roquette Freres Composition riche en methionine destinee a l'alimentation animale
CN102639691B (zh) 2009-11-30 2014-04-16 味之素株式会社 生产l-半胱氨酸的细菌以及生产l-半胱氨酸的方法
CN102782137B (zh) 2009-12-30 2016-01-06 代谢探索者公司 用于甲硫氨酸生产的菌株和方法
WO2012001003A1 (fr) 2010-07-02 2012-01-05 Metabolic Explorer Procédé de préparation d'hydroxyacides
TWI500768B (zh) 2010-07-05 2015-09-21 Metabolic Explorer Sa 由蔗糖製備１，３－丙二醇之方法
WO2012014228A1 (fr) 2010-07-28 2012-02-02 Abhishek Narain Singh Méthode pour supplanter l'activité du domaine allostérique d'une enzyme de façon à modifier sa rétroinhibition ou son activation
EP2617808B1 (fr) 2010-09-14 2016-06-15 Ajinomoto Co., Inc. Bactéries productrices d'acides aminés soufrés et procédé pour produire des acides aminés soufrés
AR083468A1 (es) 2010-10-25 2013-02-27 Metabolic Explorer Sa Aumento de la disponibilidad de nadph para la produccion de metionina
JP5847840B2 (ja) * 2010-12-30 2016-01-27 メタボリック エクスプローラー メチオニンヒドロキシ類似体（ｍｈａ）の発酵生産
WO2012090021A1 (fr) 2010-12-30 2012-07-05 Metabolic Explorer Micro-organisme recombinant pour la production par fermentation de méthionine
JP2014087259A (ja) 2011-02-22 2014-05-15 Ajinomoto Co Inc Ｌ−システイン生産菌及びｌ−システインの製造法
EP2540834A1 (fr) 2011-06-29 2013-01-02 Metabolic Explorer Procédé de préparation de 1,3-propanediol
US9410164B2 (en) 2011-10-11 2016-08-09 Metabolic Explorer Biosynthesis pathway for prenol in a recombinant microorganism
FR2983870B1 (fr) 2011-12-08 2015-07-17 Roquette Freres Composition en methionine destinee a l'alimentation animale
EP2647718A3 (fr) 2012-04-06 2014-12-24 Metabolic Explorer Procédé de production de 5-aminopentanoate en utilisant un micro-organisme recombinant
JP2012196222A (ja) * 2012-06-11 2012-10-18 Metabolic Explorer 硫酸透過酵素の発現が増強された微生物を用いてメチオニンおよびその前駆体ホモセリンまたはスクシニルホモセリンを製造するための方法
JP6100370B2 (ja) 2012-06-18 2017-03-22 メタボリック エクスプローラー メチオニンの発酵生産のための組換え微生物
WO2014049382A2 (fr) 2012-09-26 2014-04-03 Metabolic Explorer Production de fermentation d'éthylènediamine par un micro-organisme recombinant
WO2014185430A1 (fr) 2013-05-13 2014-11-20 味の素株式会社 Procédé de fabrication d'acide aminé l
PL3039153T3 (pl) 2013-08-30 2019-02-28 Evonik Degussa Gmbh Mikroorganizm do wytwarzania metioniny o ulepszonej aktywności syntazy metioniny i wypływie metioniny
JP6459962B2 (ja) 2013-10-21 2019-01-30 味の素株式会社 Ｌ−アミノ酸の製造法
EP3061828A4 (fr) 2013-10-23 2017-03-15 Ajinomoto Co., Inc. Procédé de production d'une substance cible
CN107109359B (zh) 2014-09-01 2021-06-29 赢创运营有限公司 用于通过具有改进的甲硫氨酸排出的发酵生产甲硫氨酸的方法和微生物
CN105505894A (zh) * 2014-09-24 2016-04-20 中国科学院天津工业生物技术研究所 天冬氨酸激酶/高丝氨酸脱氢酶突变体及其应用
CN107743520A (zh) * 2015-05-06 2018-02-27 特雷里斯公司 用于甲硫氨酸的生物生产的组合物和方法
EP3095868A1 (fr) 2015-05-19 2016-11-23 Evonik Degussa GmbH Production de méthionine
US20180135085A1 (en) 2015-07-10 2018-05-17 Evonik Degussa Gmbh Amino acid production
CN108026516A (zh) 2015-08-07 2018-05-11 赢创德固赛有限公司 通过发酵的蛋白硫代羧化物依赖性l-甲硫氨酸生产
BR112018010747A8 (pt) 2015-11-27 2019-02-26 Evonik Degussa Gmbh método para a produção de l-metionina
KR20180093981A (ko) 2016-01-08 2018-08-22 에보니크 데구사 게엠베하 발효적 생산에 의해 l-메티오닌을 생산하는 방법
CN105838740B (zh) * 2016-05-03 2020-11-06 湖北大学 一种茶树内生草螺菌制备纳米红色元素硒的方法
JP7066977B2 (ja) 2017-04-03 2022-05-16 味の素株式会社 Ｌ－アミノ酸の製造法
EP3856918A1 (fr) 2018-09-28 2021-08-04 Ajinomoto Co., Inc. Procédé de production de l-méthionine à l'aide d'une bactérie
WO2020071538A1 (fr) 2018-10-05 2020-04-09 Ajinomoto Co., Inc. Procédé de production d'une substance cible par fermentation bactérienne
CN112725251B (zh) * 2019-10-14 2022-07-22 江南大学 一种生产亚精胺的工程菌
CN114107243B (zh) * 2021-12-09 2023-04-07 江南大学 一种甲硫氨酸腺苷转移酶突变体
CN117535263A (zh) * 2022-11-11 2024-02-09 山东理工大学 一种基于297位点的s-腺苷蛋氨酸合成酶突变体及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000139471A (ja) 1998-11-17 2000-05-23 Ajinomoto Co Inc 発酵法によるｌ−メチオニンの製造法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4622111B2 (ja) * 2001-02-09 2011-02-02 味の素株式会社 Ｌ−システイン生産菌及びｌ−システインの製造法
DE10247437A1 (de) * 2002-10-11 2004-04-29 Consortium für elektrochemische Industrie GmbH Feedback-resistente Homoserin-Transsuccinylasen
DE10249642A1 (de) * 2002-10-24 2004-05-13 Consortium für elektrochemische Industrie GmbH Feedback-resistente Homoserin-Transsuccinylasen mit modifiziertem C-Terminus

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000139471A (ja) 1998-11-17 2000-05-23 Ajinomoto Co Inc 発酵法によるｌ−メチオニンの製造法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Molecular Cloning: a Laboratory Manual,. 2nd ed.", 1989, COLD SPRING HARBOR LAB.
KUMAR, D.; GARG, S.; BISARIA V.S.; SREEKRISHNAN, T.R.; GOMES, J.: "Production of methionine by a multi-analogue resistant mutant of Corynebacterium lilium", PROCESS BIOCHEMISTRY, vol. 38, 2003, pages 1165 - 1171
SAMBROOK ET AL.: "Molecular Cloning: a Laboratory Manual. 2nd ed.", 1989, COLD SPRING HARBOR LAB.

Cited By (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8017362B2 (en) 1998-11-17 2011-09-13 Ajinomoto Co., Inc. Method for producing L-methionine by fermentation
CN101351558B (zh) * 2006-01-04 2013-08-14 代谢探索者公司 使用硫酸盐通透酶表达增强的微生物制备甲硫氨酸及其前体高丝氨酸或琥珀酰高丝氨酸的方法
EP2314710A3 (fr) * 2006-01-04 2012-02-22 Metabolic Explorer Procédé de production de méthionine par la culture de micro-organismes modifiés pour améliorer la production de cystéine
EP1969130B1 (fr) * 2006-01-04 2014-03-12 Metabolic Explorer Procédé de synthèse de la méthionine et de ses précurseurs, l homosérine ou la succinylhomosérine, en employant un micro-organisme
US8426171B2 (en) 2006-07-28 2013-04-23 Cj Cheiljedang Corporation Microorganism producing L-methionine precursor and method of producing L-methionine and organic acid from the L-methionine precursor
WO2008013432A1 (fr) 2006-07-28 2008-01-31 Cj Cheiljedang Corporation Micro-organisme produisant un précurseur de l-méthionine et procédé de production de l-méthionine et d'acide organique à partir du précurseur de l-méthionine
JP2012213401A (ja) * 2006-07-28 2012-11-08 Cj Cheiljedang Corp Ｌ−メチオニン前駆体産生菌株
EP2657345A1 (fr) 2006-07-28 2013-10-30 CJ Cheiljedang Corporation Procédé enzymatique pour la production de L-méthionine à partir d'un précurseur et de méthylmercaptan
CN101356281B (zh) * 2006-07-28 2015-08-19 Cj第一制糖株式会社 生产l-蛋氨酸前体的微生物以及由l-蛋氨酸前体制备l-蛋氨酸和有机酸的方法
US9029105B2 (en) 2006-07-28 2015-05-12 Cj Cheiljedang Corporation Microorganism producing L-methionine precursor and method of producing L-methionine and organic acid from the L-methionine precursor
WO2008034338A1 (fr) * 2006-08-31 2008-03-27 Bioright Worldwide Company Limited Mutants de la s-adénosylméthionine synthétase, les adn les codant et les utilisations de ces mutants
US7858351B2 (en) 2006-08-31 2010-12-28 Geneharbor ( Hong Kong) Technologies, Ltd. S-adenosylmethionine synthetase mutants, the DNAs encoding the same and uses of the mutants
CN102703398B (zh) * 2007-04-11 2014-06-04 Cj第一制糖株式会社 组合物及甲硫氨酸的生产方法
CN103710320A (zh) * 2007-04-11 2014-04-09 Cj第一制糖株式会社 组合物及甲硫氨酸的生产方法
EP2808381A1 (fr) 2007-04-11 2014-12-03 CJ CheilJedang Corporation Compositions et procédés de production de méthionine
EP2808382A1 (fr) 2007-04-11 2014-12-03 CJ CheilJedang Corporation Compositions et procédés de production de méthionine
JP2010523145A (ja) * 2007-04-11 2010-07-15 シージェイ チェイルジェダン コーポレイション 組成物およびメチオニン生産方法
US9150893B2 (en) 2007-04-11 2015-10-06 Cj Cheiljedang Corporation Compositions and methods of producing methionine
CN102703398A (zh) * 2007-04-11 2012-10-03 Cj第一制糖株式会社 组合物及甲硫氨酸的生产方法
US8551742B2 (en) 2007-04-11 2013-10-08 Cj Cheiljedang Corporation Compositions and methods of producing methionine
EP2520645A2 (fr) 2007-04-11 2012-11-07 CJ CheilJedang Corporation Compositions et procédés de production de méthionine
CN103710319B (zh) * 2007-04-11 2015-10-07 Cj第一制糖株式会社 组合物及甲硫氨酸的生产方法
EP2520645A3 (fr) * 2007-04-11 2013-02-20 CJ CheilJedang Corporation Compositions et procédés de production de méthionine
US9493801B2 (en) 2007-04-11 2016-11-15 Cj Cheiljedang Corporation Compositions and methods of producing methionine
WO2008127240A1 (fr) * 2007-04-11 2008-10-23 Cargill, Incorporated Compositions et méthodes de production de méthionine
EP2573189A1 (fr) 2007-10-02 2013-03-27 Metabolic Explorer Augmentation du rendement de la méthionine
US7851180B2 (en) 2008-04-04 2010-12-14 Cj Cheiljedang Corporation Microorganism producing L-methionine precursor and the method of producing L-methionine precursor using the microorganism
EP2108693A2 (fr) 2008-04-04 2009-10-14 CJ Cheiljedang Corporation Micro-organisme produisant un précurseur de méthionine L et procédé de production du précurseur de méthionine L utilisant le microorganisme
EP3081649A1 (fr) 2008-04-04 2016-10-19 CJ CheilJedang Corporation Micro-organisme produisant un précurseur de méthionine l et procédé de production du précurseur de méthionine l utilisant le microorganisme
JP2009247354A (ja) * 2008-04-04 2009-10-29 Cj Cheiljedang Corp Ｌ−メチオニン前駆体生産菌株およびこれを用いたｌ−メチオニン前駆体の生産方法
EP2108693A3 (fr) * 2008-04-04 2009-11-11 CJ Cheiljedang Corporation Micro-organisme produisant un précurseur de méthionine L et procédé de production du précurseur de méthionine L utilisant le microorganisme
EP2128264A1 (fr) 2008-04-04 2009-12-02 CJ Cheiljedang Corporation Micro-organisme produisant un précurseur de méthionine L et procédé de production du précurseur de méthionine L utilisant le microorganisme
US8465952B2 (en) 2008-04-04 2013-06-18 Cj Cheiljedang Corporation Microorganism producing L-methionine precursor and the method of producing L-methionine precursor using the microorganism
JP2010000075A (ja) * 2008-04-04 2010-01-07 Cj Cheiljedang Corp Ｌ−メチオニン前駆体生産菌株およびこれを用いたｌ−メチオニン前駆体の生産方法
US9005952B2 (en) 2008-04-04 2015-04-14 Cj Cheiljedang Corporation Microorganism producing L-methionine precursor and the method of producing L-methionine precursor using the microorganism
CN103756948A (zh) * 2009-08-28 2014-04-30 Cj第一制糖株式会社 产生o-乙酰高丝氨酸的微生物和利用所述微生物产生o-乙酰高丝氨酸的方法
CN102002472A (zh) * 2009-08-28 2011-04-06 Cj第一制糖株式会社 产生o-乙酰高丝氨酸的微生物和利用所述微生物产生o-乙酰高丝氨酸的方法
CN102002473A (zh) * 2009-08-28 2011-04-06 Cj第一制糖株式会社 产生o-乙酰高丝氨酸的微生物和利用所述微生物产生o-乙酰高丝氨酸的方法
US8609396B2 (en) 2009-08-28 2013-12-17 Cj Cheiljedang Corporation Microorganism producing O-acetyl-homoserine and the method of producing O-acetyl-homoserine using the microorganism
WO2011073122A1 (fr) 2009-12-14 2011-06-23 Metabolic Explorer Utilisation de promoteurs inductibles dans la production de méthionine
WO2011080542A1 (fr) 2009-12-30 2011-07-07 Metabolic Explorer Augmentation de la production de méthionine par surexpression de succinate déshydrogénase
WO2012087039A2 (fr) 2010-12-21 2012-06-28 씨제이제일제당 (주) Polypeptide de variant possédant une activité d'homosérine acétyltransférase et microorganisme l'exprimant
US9234223B2 (en) 2011-04-01 2016-01-12 Ajinomoto Co., Inc. Method for producing L-cysteine
WO2012137689A1 (fr) 2011-04-01 2012-10-11 味の素株式会社 Procédé pour la production de l-cystéine
US9850514B2 (en) 2012-09-19 2017-12-26 Genentech, Inc. Methods and compositions for preventing norleucine misincorporation into proteins
US10179925B2 (en) 2012-09-19 2019-01-15 Genentech, Inc. Compositions for preventing norleucine misincorporation into proteins
US10421984B2 (en) 2012-09-19 2019-09-24 Genentech, Inc. Methods for reducing norleucine misincorporation into proteins using a microorganism comprising a mutant metA allele
US11015214B2 (en) 2012-09-19 2021-05-25 Genentech, Inc. Microorganisms comprising a mutant metA allele for reducing norleucine misincorporation into proteins
WO2018007565A1 (fr) 2016-07-08 2018-01-11 Metabolic Explorer Procédé de production fermentative de méthionine ou de sa forme analogue hydroxy par des micro-organismes comprenant des gènes codant un système de phosphotransférase de sucre (pts)
EP3296404A1 (fr) 2016-09-15 2018-03-21 Evonik Degussa GmbH Micro-organisme modifie pour production de methionine

Also Published As

Publication number Publication date
MXPA06013125A (es) 2007-02-14
PL1747269T3 (pl) 2015-08-31
JP2007536921A (ja) 2007-12-20
US8795990B2 (en) 2014-08-05
US20090029424A1 (en) 2009-01-29
ES2535816T3 (es) 2015-05-18
CN1954067B (zh) 2011-08-03
WO2005111202A1 (fr) 2005-11-24
CN1954067A (zh) 2007-04-25
EP1747269B1 (fr) 2015-03-25
EP1747269A2 (fr) 2007-01-31
WO2005108561A3 (fr) 2006-07-20
JP5164566B2 (ja) 2013-03-21

Similar Documents

Publication Publication Date Title
US8795990B2 (en) Recombinant enzyme with altered feedback sensitivity
EP2314710B1 (fr) Procédé de production de méthionine par la culture de micro-organismes modifiés pour améliorer la production de cystéine
EP2326724B1 (fr) Production de méthionine sans méthionine n-acylé
US20080286840A1 (en) Microorganisms Comprising Enzymes Express with Low Gamma-Elimination Activity
US9506093B2 (en) Recombinant microorganism for the fermentative production of methionine
JP5944323B2 (ja) メチオニンの生産における誘導プロモーターの使用
US20120288904A1 (en) Strains and method for the production of methionine
EP1846564A2 (fr) Procede destine a la production enzymatique d'alpha-cetobutyrate
BRPI0510819B1 (pt) método para a preparação de metionina, seus precursores ou produtos derivados da mesma e microorganismo recombinante
JP2012196222A (ja) 硫酸透過酵素の発現が増強された微生物を用いてメチオニンおよびその前駆体ホモセリンまたはスクシニルホモセリンを製造するための方法
MX2008008777A (en) Process for the preparation of methionine and its precursors homoserine or succinylhomoserine employing a microorganism with enhanced sulfate permease expression

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 6007/DELNP/2006

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: 2005742717

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2007512216

Country of ref document: JP

Ref document number: PA/A/2006/013125

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 200580015315.2

Country of ref document: CN

NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Ref document number: DE

WWE Wipo information: entry into national phase

Ref document number: 11579907

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 2005742717

Country of ref document: EP

ENP Entry into the national phase

Ref document number: PI0510819

Country of ref document: BR